Nanoparticles have been a subject of great interest, promising extensive applications including display devices, information storage, biological tagging materials, diagnostic imaging, drug delivery, theranostics, photovoltaics, sensors and catalysts. Nanoparticles having small diameters can have properties intermediate between molecular and bulk forms of matter. For example, nanoparticles based on semiconductor materials having small diameters can exhibit quantum confinement of both the electron and hole in all three dimensions, which leads to an increase in the effective band gap of the material with decreasing crystallite size. Moreover, the magnetic properties of matter change dramatically depending on the particle size. Likewise, the plasmon-related absorption of nanoparticles, e.g. gold nanoparticles largely depends on the size and the shape of said particles. Biological effects of nanoparticles, e.g. permeability through biological barriers, like membranes, skin etc. or toxicity show distinct size-related dependencies. Most nanoparticles consist of an inorganic core that is surrounded by a layer of organic ligands. This organic ligand shell is critical to the nanoparticles for controlled growth, stability, prevention of agglomeration, processing, modification, and incorporation into various substrates. Originally, this organic ligand shell around a nanoparticle is formed in situ after nucleation and growth of the nanoparticle in its growth solution. For most applications, nanoparticles must be processed outside of their growth solution and transferred into various chemical environments. However, nanoparticles often lose their specific physical and chemical properties, e.g. high fluorescence, or become irreversibly aggregated when removed from their growth solution. Commonly, nanoparticles of defined shape, size and physico-chemical properties are synthesized in high boiling hydrophobic solvents, e.g. diphenyl ether, squalene and the like, containing hydrophobic ligands, e.g. trialkyl phosphines or phosphine oxides, e.g. trioctyl phosphine (TOP) or its corresponding phosphine oxide (TOPO), long chain aliphatic or unsaturated fatty acids or amines, e.g. oleic acid (OA) or hexadecyl amine (HDA) and the like, which—albeit indispensable for the synthesis-restrict the use of nanoparticles in diverse technical fields mentioned above. Different methods have been described for the transfer of nanoparticles into various chemical environments, e.g. ligand exchange or encapsulation into polymer micelles or polymersomes.
A micelle comprises of a plurality of amphiphilic molecules, each consisting of a hydrophilic “head” region and a hydrophobic “tail” region in the same molecule. Said amphiphilic molecule may be a small molecule with a low molecular weight, typically below 1000 Dalton, e.g. sodium dodecyl sulfate, salts of long chain fatty acids, phospholipids, and the like, or an amphiphilic block copolymer. Hence, it should be noted that in the context of the invention, the amphiphilic subunits a micelle is comprised of are termed as “unimers”, regardless of the molecular weight or the amphiphilic chemical structure. Thus the terms “unimer” or “unimers” will be applied to amphiphilic block polymers as well, if molecules of said polymers account for the amphiphilic subunits of a micelle.
In general, micellization, i.e. micelle formation of amphiphilic molecules takes place either in solvents like water or in unpolar organic solvents, e.g. hydrocarbons, e.g. hexane, toluene and the like. In the first case a normal micelle, in the latter an inverse (or reverse) micelle is formed. The terms “reverse micelle” and “inverse micelle” are used synonymously within the scope of the invention. This self-assembly process relies on a thermodynamic equilibrium. In an aqueous environment solvation of the hydrophobic tail is entropically unfavorable. Nonetheless, at very low concentrations the unimers are still completely dissolved, i.e. in a disordered highly entropic condition. With increasing concentrations of the unimer the hydrophobic tails sequester away from the water phase by micelle formation, i.e. forming ordered, mostly—but not exclusively-spherical arrangements comprised of a hydrophobic core and a hydrophilic shell, namely a normal micelle. The entropy gained from the release of water molecules from the solvation shell of the hydrophobic tails outweighs the loss of entropy caused by micelle formation. The minimum concentration of unimers at which micelle formation starts to happen is the so-called critical micelle concentration (CMC)
Reversely, in a non-polar organic solvent, an inverse micelle is formed, wherein the hydrophilic head groups of the amphiphilic unimers aggregate to a hydrophilic core, shielded from the non-polar solvent by a shell of hydrophobic tails.
Accordingly, a micelle and its corresponding dissolved unimers are in a dynamic equilibrium, thus the stability of micelles depends on the concentration of unimers, salt concentration, temperature etc. This thermodynamic equilibrium can be frozen in favor of the micelle formation, if the unimers of which the micelle is comprised of, are irreversibly cross-linked.
It has been demonstrated that in an aqueous environment hydrophobic nanoparticles, as mentioned above, can be enclosed into the hydrophobic core of a micelle, when mixed with amphiphilic molecules, hence producing an aqueous colloidal solution of said nanoparticles. (E. Pöselt, S. Fischer, S. Förster, H. Weller; Highly Stable Biocompatible Inorganic Nanoparticles by Self-Assembly of Triblock-Copolymer Ligands Langmuir 2009, 25, 13906-13913.) (B.-S. Kim, J.-M. Qiu, J.-P. Wang, T. A. Taton; Magnetomicelles: Composite Nanostructures from Magnetic Nanoparticles and Cross-Linked Amphiphilic Block Copolymers Nano Letters 2005, 5, 1987-1991.) (E. Pöselt, C. Schmidtke, S. Fischer, K. Peldschus, J. Salamon, H. Kloust, H. Tran, A. Pietsch, M. Heine, G. Adam, U. Schumacher, C. Wagener, S. Förster, H. Weller; Tailor-Made Quantum Dot and Iron Oxide Based Contrast Agents for in Vitro and in Vivo Tumor Imaging ACS Nano 2012, 6, 3346-3355.) (C.-A. J. Lin, R. A. Sperling, J. K. Li, T.-Y. Yang, P.-Y. Li, M. Zanella, W. H. Chang, W. J. Parak, Design of an Amphiphilic Polymer for Nanoparticle Coating and Functionalization Small 2008, 3, 334-341.) Said amphiphilic molecules include naturally occurring lipids, e.g. phospholipids like lecithin, cationic, zwitterionic, anionic and non-ionic surfactants, as well as macromolecular amphiphiles like block copolymers, comprised of at least two polymer blocks of different compatibility to a given solvent, according to the Flory-Huggins theory, e.g. Thermodynamic Properties of Solutions of Long-Chain Compounds, Annals of the New York Academy of Sciences, Volume 43, p. 1-32 (1942); Paul J. Flory, Thermodynamics of High Polymer Solutions; The Journal of Chemical Physics 10, 51 (1942).
It has also been demonstrated that hydrophilic nanocrystals can be grown in the interior volume of reverse micelles. However, the quality of the nanocrystals generated by this method is often unsatisfying with respect to their properties.
It is evident that said aqueous colloidal preparation of micellar enclosed nanoparticles determines and restricts the use of this preparation. Albeit eventually suitable for biological or medical applications, said aqueous preparations show a number of shortcomings. Firstly, the transfer of fluorescent semi conductor nanoparticles into water may deteriorate their fluorescence properties by quenching. In another aspect, the coupling chemistry for biofunctionalization, i.e. the conjugation of micellar encapsulated nanoparticles with biofunctional entities, like proteins, peptides, antibodies, carbohydrates, lectins, nucleic acid fragments, DNA, RNA, aptamers etc., often benefits from non-aqueous conditions. The aqueous environment may hamper conjugation of said entities, because the conjugation site is poorly accessible, due to folding and coiling of said entities in an aqueous environment. In contrast, many of said biofunctional entities disentangle under non aqueous conditions, thus making putative coupling sites well amenable to conjugation. Consequently, aqueous compositions of nanoparticles cannot be used under said conditions, or suffer from strongly reduced coupling yields.
Furthermore, regiochemistry of the coupling substrate may be ambiguous, due to a plurality of functionalities of similar reactivity, e.g. multiple lysine residues, hence leading to intractable mixtures of specimens of undefined biological activity. In principle, this drawback can be avoided by employing suitable protective group chemistry, in order to control regiochemistry. However, protection of proteins, peptides, antibodies, carbohydrates, lectins, nucleic acid fragments, DNA, RNA, aptamers etc. frequently renders these molecules sparingly soluble or even insoluble in water, making the coupling conditions incompatible to aqueous compositions of micelle encapsulated nanoparticles.
Moreover, said biofunctionalized nanoparticle conjugates require low storage temperatures in order to prevent decomposition. Unfortunately, the matrix conditions in frozen aqueous solutions at or below 0° C. are often detrimental to the physicochemical properties of nanoparticles, e.g. causing loss of fluorescence or irreversible aggregation after thawing.
Decomposition of said biofunctionalized nanoparticle conjugates may also occur from microbial infestation. Therefore, an addition of microbicides, e.g. sodium azide is frequently required. However, said additives are frequently detrimental to common coupling reactions and furthermore often interfere with or even override the intrinsic biological activities of said biofunctionalized nanoparticle conjugates, thus distorting testing results.
It is therefore an object of the present invention to resolve said shortcomings and to provide an improved composition comprising micelles, which encapsulate nanoparticles and to provide an improved use of such a composition and methods for providing such a composition.